Multiparametric flow cytometry of T-cell subsets

We used flow cytometry analysis to assess the production of FOXP3, Helios, GATA3, IL-9, IL-17A, and IL-10 by CXCR6+ and CD4+ T cells. Briefly, splenocytes were incubated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Sigma-Aldrich) for 4 h in the presence of brefeldin-A (GolgiPlug, BD Biosciences), which prevents the transport of cytokines and transcription factors out of the cell [12 (link),33 (link),41 (link)]. Cells were washed and surface stained for CD4, and CXCR6 surface receptors (BioLegend, San Diego, CA, USA). After permeabilization and fixation (BioLegend), the cells were stained with intracellular cytokines (anti-IL-9, anti-IL-10, and anti-IL-17A; BioLegend) and transcription factors (anti-FOXP3, anti-GATA3, and anti-Helios; BioLegend). The proportions of CXCR6+FOXP3+, CXCR6+Helios+, CD4+GATA3+, CD4+IL-9+, CXCR6+IL-10+, and CD4+IL-17A+ cells were acquired via a FC 500 flow cytometer and analyzed using CXP software (Beckman Coulter, Indianapolis, IN, USA).